An intracellular 6G-fructofuranosidase (endo-type enzyme) extracted from Xanthophyllomyces dendrorhous 269 efficiently hydrolyzes fructosyl-β-(2→1)-linked sucrose to produce neo-kestose as a main transglycosylation product. The enzyme with a molecular weight of 33 kDa was purified by DEAE-52 cellulose chromatography. Thirty-fivefold purification and a 13.4% enzyme activity recovery were achieved. Optimum enzyme activity occurred at pH 6.4 and 45 °C and the enzyme was stable at pH 4-7 and at 45 °C. Using sucrose as a substrate, the Km and Vmax values were, respectively, 511 mmol/l and 233 μmol/(min mg) for transfer activity and 62 mmol/l and 164 μmol/(min mg) for hydrolytic activity. Under optimum conditions, a maximum concentration (73.9 g/l) of neo-fructooligosaccharides catalyzed by the endo-enzyme was obtained. These findings suggest that the purified endo-enzyme exhibits a high transfructosylation activity and it has potential for the industrial production of neo-FOSs.